Labelled nucleotides

ABSTRACT

Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 12/460,741, filed Jul. 23, 2009, now U.S. Pat. No. 7,785,796, whichis a Divisional Application of U.S. application Ser. No. 11/301,578,filed Dec. 13, 2005, now U.S. Pat. No. 7,566,537, which is a DivisionalApplication of U.S. application Ser. No. 10/227,131, filed Aug. 23,2002, now U.S. Pat. No. 7,057,026, which in turn claims benefit ofUnited Kingdom Application No. GB0129012.1, filed Dec. 4, 2001. Theentire teachings of the above applications are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to labelled nucleotides. In particular, thisinvention discloses nucleotides having a removable label and their usein polynucleotide sequencing methods.

BACKGROUND

Advances in the study of molecules have been led, in part, byimprovement in technologies used to characterise the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis and the study of hybridisation events.

An example of the technologies that have improved the study of nucleicacids, is the development of fabricated arrays of immobilised nucleicacids. These arrays consist typically of a high-density matrix ofpolynucleotides immobilised onto a solid support material. See, e.g.,Fodor et al., Trends Biotech. 12:19-26, 1994, which describes ways ofassembling the nucleic acids using a chemically sensitized glass surfaceprotected by a mask, but exposed at defined areas to allow attachment ofsuitably modified nucleotide phosphoramidites. Fabricated arrays canalso be manufactured by the technique of “spotting” knownpolynucleotides onto a solid support at predetermined positions (e.g.,Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383, 1995).

A further development in array technology is the attachment of thepolynucleotides to the solid support material to form single moleculearrays. Arrays of this type are disclosed in International Patent App.WO 00/06770. The advantage of these arrays is that reactions can bemonitored at the single molecule level and information on large numbersof single molecules can be collated from a single reaction.

For DNA arrays to be useful, the sequences of the molecules must bedetermined. U.S. Pat. No. 5,302,509 discloses a method to sequencepolynucleotides immobilised on a solid support. The method relies on theincorporation of 3′-blocked bases A, G, C and T having a differentfluorescent label to the immobilised polynucleotide, in the presence ofDNA polymerase. The polymerase incorporates a base complementary to thetarget polynucleotide, but is prevented from further addition by the3′-blocking group. The label of the incorporated base can then bedetermined and the blocking group removed by chemical cleavage to allowfurther polymerisation to occur.

Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) describes the synthesisof nucleotide triphosphates modified with a 3′-O-blocking group that isphotolabile and fluorescent. The modified nucleotides are intended foruse in DNA sequencing experiments. However, these nucleotides proved tobe difficult to incorporate onto an existing polynucleotide, due to aninability to fit into the polymerase enzyme active site.

Zhu et al. (Cytometry 28:206-211, 1997) also discloses the use offluorescent labels attached to a nucleotide via the base group. Thelabelled nucleotides are intended for use in fluorescence in situhybridisation (FISH) experiments, where a series of incorporatedlabelled nucleotides is required to produce a fluorescent “bar code”.

SUMMARY OF THE INVENTION

In the present invention, a nucleoside or nucleotide molecule is linkedto a detectable label via a cleavable linker group attached to the base,rendering the molecule useful in techniques using Labelled nucleosidesor nucleotides, e.g., sequencing reactions, polynucleotide synthesis,nucleic acid amplification, nucleic acid hybridization assays, singlenucleotide polymorphism studies, and other techniques using enzymes suchas polymerases, reverse transcriptases, terminal transferases, or otherDNA modifying enzymes. The invention is especially useful in techniquesthat use Labelled dNTPs, such as nick translation, random primerlabeling, end-labeling (e.g., with terminaldeoxynucleotidyltransferase), reverse transcription, or nucleic acidamplification. The molecules of the present invention are in contrast tothe prior art, where the label is attached to the ribose or deoxyribosesugar, or where the label is attached via a non-cleavable linker.

According to a first aspect of the invention, a nucleotide or nucleosidemolecule, or an analog thereof, has a base that is linked to adetectable label via a cleavable linker.

The invention features a nucleotide or nucleoside molecule, having abase that is linked to a detectable label via a cleavable linker. Thebase can be a purine, or a pyrimidine. The base can be a deazapurine.The molecule can have a ribose or deoxyribose sugar moiety. The riboseor deoxyribose sugar can include a protecting group attached via the 2′or 3′ oxygen atom. The protecting group can be removed to expose a3′-OH. The molecule can be a deoxyribonucleotide triphosphate. Thedetectable label can be a fluorophore. The linker can be an acid labilelinker, a photolabile linker, or can contain a disulphide linkage.

The invention also features a method of labeling a nucleic acidmolecule, where the method includes incorporating into the nucleic acidmolecule a nucleotide or nucleoside molecule, where the nucleotide ornucleoside molecule has a base that is linked to a detectable label viaa cleavable linker. The incorporating step can be accomplished via aterminal transferase, a polymerase or a reverse transcriptase. The basecan be a purine, or a pyrimidine. The base can be a deazapurine. Thenucleotide or nucleoside molecule can have a ribose or deoxyribose sugarmoiety. The ribose or deoxyribose sugar can include a protecting groupattached via the 2′ or 3′ oxygen atom. The protecting group can beremoved to expose a 3′-OH group. The molecule can be adeoxyribonucleotide triphosphate. The detectable label can be afluorophore. The linker can be an acid labile linker, a photolabilelinker, or can contain a disulphide linkage. The detectable label and/orthe cleavable linker can be of a size sufficient to prevent theincorporation of a second nucleotide or nucleoside into the nucleic acidmolecule.

In another aspect, the invention features a method for determining thesequence of a target single-stranded polynucleotide, where the methodincludes monitoring the sequential incorporation of complementarynucleotides, where the nucleotides each have a base that is linked to adetectable label via a cleavable linker, and where the identity of eachnucleotide incorporated is determined by detection of the label linkedto the base, and subsequent removal of the label.

The invention also features a method for determining the sequence of atarget single-stranded polynucleotide, where the method includes: (a)providing nucleotides, where the nucleotides have a base that is linkedto a detectable label via a cleavable linker, and where the detectablelabel linked to each type of nucleotide can be distinguished upondetection from the detectable label used for other types of nucleotides;(b) incorporating a nucleotide into the complement of the target singlestranded polynucleotide; (c) detecting the label of the nucleotide of(b), thereby determining the type of nucleotide incorporated; (d)removing the label of the nucleotide of (b); and (e) optionallyrepeating steps (b)-(d) one or more times; thereby determining thesequence of a target single-stranded polynucleotide.

In the methods described herein, each of the nucleotides can be broughtinto contact with thew target sequentially, with removal ofnon-incorporated nucleotides prior to addition of the next nucleotide,where detection and removal of the label is carried out either afteraddition of each nucleotide, or after addition of all four nucleotides.

In the methods, all of the nucleotides can be brought into contact withthe target simultaneously, i.e., a composition comprising all of thedifferent nucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label(s).

The methods can comprise a first step and a second step, where in thefirst step, a first composition comprising two of the four nucleotidesis brought into contact with the target, and non-incorporatednucleotides are removed prior to detection and subsequent to removal ofthe label, and where in the second step, a second composition comprisingthe two nucleotides not included in the first composition is broughtinto contact with the target, and non-incorporated nucleotides areremoved prior to detection and subsequent to removal of the label, andwhere the first steps and the second step can be optionally repeated oneor more times.

The methods described herein can also comprise a first step and a secondstep, where in the first step, a composition comprising one of the fournucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label, and where in the second step, asecond composition comprising the three nucleotides not included in thefirst composition is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label, and where the first steps and thesecond step can be optionally repeated one or more times.

The methods described herein can also comprise a first step and a secondstep, where in the first step, a first composition comprising three ofthe four nucleotides is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label, and where in the second step, acomposition comprising the nucleotide not included in the firstcomposition is brought into contact with the target, andnon-incorporated nucleotides are removed prior to detection andsubsequent to removal of the label, and where the first steps and thesecond step can be optionally repeated one or more times.

In a further aspect, the invention features a kit, where the kitincludes: (a) individual the nucleotides, where each nucleotide has abase that is linked to a detectable label via a cleavable linker, andwhere the detectable label linked to each nucleotide can bedistinguished upon detection from the detectable label used for otherthree nucleotides; and (b) packaging materials therefor. The kit canfurther include an enzyme and buffers appropriate for the action of theenzyme.

The nucleotides/nucleosides are suitable for use in many differentDNA-based methodologies, including DNA synthesis and DNA sequencingprotocols.

According to another aspect of the invention, a method for determiningthe sequence of a target polynucleotide comprises monitoring thesequential incorporation of complementary nucleotides, wherein thenucleotides comprise a detectable label linked to the base portion ofthe nucleotide via a cleavable linker, incorporation is detected bymonitoring the label, and the label is removed to permit furthernucleotide incorporation to occur.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary nucleotide structures useful in the invention.For each structure, X can be H, phosphate, diphosphate or triphosphate.R₁ and R₂ can be the same or different, and can be selected from H, OH,or any group which can be transformed into an OH, including, but notlimited to, a carbonyl. Some suitable functional groups for R₁ and R₂include the structures shown in FIG. 3.

FIG. 2 shows structures of linkers useful in the invention, including(1) disulfide linkers and acid labile linkers, (2) dialkoxybenzyllinkers, (3) Sieber linkers, (4) indole linkers and (5) t-butyl Sieberlinkers.

FIG. 3 shows some functional molecules useful in the invention,including some cleavable linkers and some suitable hydroxyl protectinggroups. In these structures, R₁ and R₂ may be the same of different, andcan be H, OH, or any group which can be transformed into an OH group,including a carbonyl. R₃ represents one or more substituentsindependently selected from alkyl, alkoxyl, amino or halogen groups.Alternatively, cleavable linkers may be constructed from any labilefunctionality used on the 3′-block. R₄ and R₅ can be H or alkyl, and R₆can be alkyl, cycloalkyl, alkenyl, cycloalkenyl or benzyl. X can be H,phosphate, diphosphate or triphosphate.

FIG. 4 shows a denaturing gel showing the incorporation of thetriphosphate of Example 1 using Klenow polymerase.

FIG. 5 shows a denaturing gel showing the incorporation of thetriphosphate of Example 3 using Klenow polymerase.

FIG. 6 shows a denaturing gel showing the incorporation of thetriphosphate of Example 4 using Klenow polymerase.

DETAILED DESCRIPTION

The present invention relates to nucleotides and nucleosides that aremodified by attachment of a label via a cleavable linker, therebyrendering the molecule useful in techniques where the labelled moleculeis to interact with an enzyme, such as sequencing reactions,polynucleotide synthesis, nucleic acid amplification, nucleic acidhybridization assays, single nucleotide polymorphism studies, techniquesusing enzymes such as polymerase, reverse transcriptase, terminaltransferase, techniques that use Labelled dNTPs (e.g., nick translation,random primer labeling, end-labeling (e.g., with terminaldeoxynucleotidyltransferase), reverse transcription, or nucleic acidamplification).

As is known in the art, a “nucleotide” consists of a nitrogenous base, asugar, and one or more phosphate groups. In RNA, the sugar is a ribose,and in DNA is a deoxyribose, i.e., a sugar lacking a hydroxyl group thatis present in ribose. The nitrogenous base is a derivative of purine orpyrimidine. The purines are adenosine (A) and guanidine (G), and thepyrimidines are cytidine (C) and thymidine (T) (or in the context ofRNA, uracil (U)). The C-1 atom of deoxyribose is bonded to N-1 of apyrimidine or N-9 of a purine. A nucleotide is also a phosphate ester ofa nucleoside, with esterification occurring on the hydroxyl groupattached to C-5 of the sugar. Nucleotides are usually mono, di- ortriphosphates.

A “nucleoside” is structurally similar to a nucleotide, but are missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogs areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analog” means a compound or molecule whose core structure is the sameas, or closely resembles that of, a parent compound, but which has achemical or physical modification, such as a different or additionalside groups, which allows the derivative nucleotide or nucleoside to belinked to another molecule. For example, the base can be a deazapurine.The derivatives should be capable of undergoing Watson-Crick pairing.“Deivative” and “analog” also mean a synthetic nucleotide or nucleosidederivative having modified base moieties and/or modified sugar moieties.Such derivatives and analogs are discussed in, e.g., Scheit, NucleotideAnalogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews90:543-584, 1990. Nucleotide analogs can also comprise modifiedphosphodiester linkages, including phosphorothioate, phosphorodithioate,alkylphosphonate, phosphoranilidate and phosphoramidate linkages. Theanalogs should be capable of undergoing Watson-Crick base pairing.“Derivative” and “analog”, as used herein, may be used interchangeably,and are encompassed by the terms “nucleotide” and “nucleoside” asdefined herein.

The present invention can make use of conventional detectable labels.Detection can be carried out by any suitable method, includingfluorescence spectroscopy or by other optical means. The preferred labelis a fluorophore, which, after absorption of energy, emits radiation ata defined wavelength. Many suitable fluorescent labels are known. Forexample, Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) disclosesdansyl-functionalised fluorescent moieties that can be used in thepresent invention. Zhu et al. (Cytometry 28:206-211, 1997) describes theuse of the fluorescent labels Cy3 and Cy5, which can also be used in thepresent invention. Labels suitable for use are also disclosed in Proberet al. (Science 238:336-341, 1987); Connell et al. (BioTechniques5(4):342-384, 1987), Ansorge et al. (Nucl. Acids. Res. 15(11):4593-4602,1987) and Smith et al. (Nature 321:674, 1986). Other commerciallyavailable fluorescent labels include, but are not limited to,fluorescein, rhodamine (including TMR, texas red and Rox), alexa,bodipy, acridine, coumarin, pyrene, benzanthracene and the cyanins.

Multiple labels can also be used in the invention. For example,bi-fluorophore FRET cassettes (Tet. Letts. 46:8867-8871, 2000) are wellknown in the art and can be utilised in the present invention.Multi-fluor dendrimeric systems (J. Amer. Chem. Soc. 123:8101-8108,2001) can also be used.

Although fluorescent labels are preferred, other forms of detectablelabels will be apparent as useful to those of ordinary skill. Forexample, microparticles, including quantum dots (Empodocles, et al.,Nature 399:126-130, 1999), gold nanoparticles (Reichert et al., Anal.Chem. 72:6025-6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad.Sci. USA 97 (17):9461-9466, 2000), and tags detectable by massspectrometry can all be used.

Multi-component labels can also be used in the invention. Amulti-component label is one which is dependent on the interaction witha further compound for detection. The most common multi-component labelused in biology is the biotin-streptavidin system. Biotin is used as thelabel attached to the nucleotide base. Streptavidin is then addedseparately to enable detection to occur. Other multi-component systemsare available. For example, dinitrophenol has a commercially availablefluorescent antibody that can be used for detection.

The label (or label and linker construct) can be of a size or structuresufficient to act as a block to the incorporation of a furthernucleotide onto the nucleotide of the invention. This permits controlledpolymerization to be carried out. The block can be due to sterichindrance, or can be due to a combination of size, charge and structure.

The invention will be further described with reference to nucleotides.However, unless indicated otherwise, the reference to nucleotides isalso intended to be applicable to nucleosides. The invention will alsobe further described with reference to DNA, although the descriptionwill also be applicable to RNA, PNA, and other nucleic acids, unlessotherwise indicated.

The modified nucleotides of the invention use a cleavable linker toattach the label to the nucleotide. The use of a cleavable linkerensures that the label can, if required, be removed after detection,avoiding any interfering signal with any labelled nucleotideincorporated subsequently.

Cleavable linkers are known in the art, and conventional chemistry canbe applied to attach a linker to a nucleotide base and a label. Thelinker can be cleaved by any suitable method, including exposure toacids, bases, nucleophiles, electrophiles, radicals, metals, reducing oroxidising agents, light, temperature, enzymes etc. Suitable linkers canbe adapted from standard chemical blocking groups, as disclosed inGreene & Wuts, Protective Groups in Organic Synthesis, John Wiley &Sons. Further suitable cleavable linkers used in solid-phase synthesisare disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

The use of the term “cleavable linker” is not meant to imply that thewhole linker is required to be removed from the nucleotide base. Thecleavage site can be located at a position on the linker that ensuresthat part of the linker remains attached to the nucleotide base aftercleavage.

The linker can be attached at any position on the nucleotide baseprovided that Watson-Crick base pairing can still be carried out. In thecontext of purine bases, it is preferred if the linker is attached viathe 7 position of the purine or the preferred deazapurine analogue, viaan 8-modified purine, via an N-6 modified adenosine or an N-2 modifiedguanine. For pyrimidines, attachment is preferably via the 5 position oncytidine, thymidine or uracil and the N-4 position on cytosine. Suitablenucleotide structures are shown in FIG. 1. For each structure in FIG. 1,X can be H, phosphate, diphosphate or triphosphate. R₁ and R₂ can be thesame or different, and can be selected from H, OH, or any group whichcan be transformed into an OH, including, but not limited to, acarbonyl. Some suitable functional groups for R₁ and R₂ include thestructures shown in FIG. 3.

Suitable linkers are shown in FIG. 2 and include, but are not limitedto, disulfide linkers (1), acid labile linkers (2, 3, 4 and 5; includingdialkoxybenzyl linkers (e.g., 2), Sieber linkers (e.g., 3), indolelinkers (e.g., 4), t-butyl Sieber linkers (e.g., 5)), electrophilicallycleavable linkers, nucleophilically cleavable linkers, photocleavablelinkers, cleavage under reductive conditions, oxidative conditions,cleavage via use of safety-catch linkers, and cleavage by eliminationmechanisms.

A. Electrophilically Cleaved Linkers.

Electrophilically cleaved linkers are typically cleaved by protons andinclude cleavages sensitive to acids. Suitable linkers include themodified benzylic systems such as trityl, p-alkoxybenzyl esters andp-alkoxybenzyl amides. Other suitable linkers includetert-butyloxycarbonyl (Boc) groups and the acetal system (e.g., as isshown in FIG. 3 as O—C(R₄)(R₅)—O—R₆.

The use of thiophilic metals, such as nickel, silver or mercury, in thecleavage of thioacetal or other sulphur-containing protecting groups canalso be considered for the preparation of suitable linker molecules.

B. Nucleophilically Cleaved Linkers.

Nucleophilic cleavage is also a well recognised method in thepreparation of linker molecules. Groups such as esters that are labilein water (i.e., can be cleaved simply at basic pH) and groups that arelabile to non-aqueous nucleophiles, can be used. Fluoride ions can beused to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethyl silane (TBDMS).

C. Photocleavable Linkers.

Photocleavable linkers have been used widely in carbohydrate chemistry.It is preferable that the light required to activate cleavage does notaffect the other components of the modified nucleotides. For example, ifa fluorophore is used as the label, it is preferable if this absorbslight of a different wavelength to that required to cleave the linkermolecule.

Suitable linkers include those based on O-nitrobenzyl compounds andnitroveratryl compounds. Linkers based on benzoin chemistry can also beused (Lee et al., J. Org. Chem. 64:3454-3460, 1999).

D. Cleavage Under Reductive Conditions

There are many linkers known that are susceptible to reductive cleavage.Catalytic hydrogenation using palladium-based catalysts has been used tocleave benzyl and benzyloxycarbonyl groups. Disulphide bond reduction isalso known in the art.

E. Cleavage Under Oxidative Conditions

Oxidation-based approaches are well known in the art. These includeoxidation ofp-alkoxybenzyl groups and the oxidation of sulphur andselenium linkers. The use of aqueous iodine to cleave disulphides andother sulphur or selenium-based linkers is also within the scope of theinvention.

F. Safety-Catch Linkers

Safety-catch linkers are those that cleave in two steps. In a preferredsystem the first step is the generation of a reactive nucleophiliccenter followed by a second step involving an intra-molecularcyclization that results in cleavage. For example, levulinic esterlinkages can be treated with hydrazine or photochemistry to release anactive amine, which can then be cyclised to cleave an ester elsewhere inthe molecule (Burgess et al., J. Org. Chem. 62:5165-5168, 1997).

G. Cleavage by Elimination Mechanisms

Elimination reactions can also be used. For example, the base-catalysedelimination of groups such as Fmoc and cyanoethyl, andpalladium-catalysed reductive elimination of allylic systems, can beused.

As well as the cleavage site, the linker can comprise a spacer unit. Thespacer distances the nucleotide base from the cleavage site or label.The length of the linker is unimportant provided that the label is helda sufficient distance from the nucleotide so as not to interfere withany interaction between the nucleotide and an enzyme.

The modified nucleotides can also comprise additional groups ormodifications to the sugar group. For example, a dideoxyribosederivative, lacking two oxygens on the ribose ring structure (at the 2′and 3′ positions), can be prepared and used as a block to furthernucleotide incorporation on a growing oligonucleotide strand. The ribosering can also be modified to include a protecting group at the 3′position or a group that can be transformed or modified to form a 3′ OHgroup. The protecting group is intended to prevent nucleotideincorporation onto a nascent polynucleotide strand, and can be removedunder defined conditions to allow polymerisation to occur. In contrastto the prior art, there is no detectable label attached at the ribose 3′position. This ensures that steric hindrance with the polymerase enzymeis reduced, while still allowing control of incorporation using theprotecting group.

The skilled person will appreciate how to attach a suitable protectinggroup to the ribose ring to block interactions with the 3′-OH. Theprotecting group can be attached directly at the 3′ position, or can beattached at the 2′ position (the protecting group being of sufficientsize or charge to block interactions at the 3′ position). Alternatively,the protecting group can be attached at both the 3′ and 2′ positions,and can be cleaved to expose the 3′OH group.

Suitable protecting groups will be apparent to the skilled person, andcan be formed from any suitable protecting group disclosed in Green andWuts, supra. Some examples of such protecting groups are shown in FIG.3. The protecting group should be removable (or modifiable) to produce a3′ OH group. The process used to obtain the 3′ OH group can be anysuitable chemical or enzymic reaction.

The labile linker may consist of functionality cleavable under identicalconditions to the block. This will make the deprotection process moreefficient as only a single treatment will be required to cleave both thelabel and the block. Thus the linker may contain functional groups asdescribed in FIG. 3, which could be cleaved with the hydroxylfunctionality on either the residual nucleoside or the removed label.The linker may also consist of entirely different chemical functionalitythat happens to be labile to the conditions used to cleave the block.

The term “alkyl” covers both straight chain and branched chain alkylgroups. Unless the context indicates otherwise, the term “alkyl” refersto groups having 1 to 8 carbon atoms, and typically from 1 to 6 carbonatoms, for example from 1 to 4 carbon atoms. Examples of alkyl groupsinclude methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl,n-pentyl, 2-pentyl, 3-pentyl, 2-methyl butyl, 3-methyl butyl, andn-hexyl and its isomers.

Examples of cycloalkyl groups are those having from 3 to 10 ring atoms,particular examples including those derived from cyclopropane,cyclobutane, cyclopentane, cyclohexane and cycloheptane, bicycloheptaneand decalin.

Examples of alkenyl groups include, but are not limited to,ethenyl(vinyl), 1-propenyl, 2-propenyl(allyl), isopropenyl, butenyl,buta-1,4-dienyl, pentenyl, and hexenyl.

Examples of cycloalkenyl groups include, but are not limited to,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl andcyclohexenyl.

The term alkoxy refers to C₁₋₆ alkoxy unless otherwise indicated: —OR,wherein R is a C₁₋₆alkyl group. Examples of C₁₋₆ alkoxy groups include,but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr)(n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu)(sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).

The term amino refers to groups of type NR¹R², wherein R¹ and R² areindependently selected from hydrogen, a C₁₋₆ alkyl group (also referredto as C₁₋₆ alkylamino or di-C₁₋₆ alkylamino).

The term “halogen” as used herein includes fluorine, chlorine, bromineand iodine.

The nucleotide molecules of the present invention are suitable for usein many different methods where the detection of nucleotides isrequired.

DNA sequencing methods, such as those outlined in U.S. Pat. No.5,302,509 can be carried out using the nucleotides.

A method for determining the sequence of a target polynucleotide can becarried out by contacting the target polynucleotide separately with thedifferent nucleotides to form the complement to that of the targetpolynucleotide, and detecting the incorporation of the nucleotides. Sucha method makes use of polymerisation, whereby a polymerase enzymeextends the complementary strand by incorporating the correct nucleotidecomplementary to that on the target. The polymerisation reaction alsorequires a specific primer to initiate polymerisation.

For each cycle, the incorporation of the labelled nucleotide is carriedout by the polymerase enzyme, and the incorporation event is thendetermined. Many different polymerase enzymes exist, and it will beevident to the person of ordinary skill which is most appropriate touse. Preferred enzymes include DNA polymerase I, the Klenow fragment,DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase or ventpolymerase. A polymerase engineered to have specific properties can alsobe used.

The sequencing methods are preferably carried out with the targetpolynucleotide arrayed on a solid support. Multiple targetpolynucleotides can be immobilised on the solid support through linkermolecules, or can be attached to particles, e.g., microspheres, whichcan also be attached to a solid support material.

The polynucleotides can be attached to the solid support by a number ofmeans, including the use of biotin-avidin interactions. Methods forimmobilizing polynucleotides on a solid support are well known in theart, and include lithographic techniques and “spotting” individualpolynucleotides in defined positions on a solid support. Suitable solidsupports are known in the art, and include glass slides and beads,ceramic and silicon surfaces and plastic materials. The support isusually a flat surface although microscopic beads (microspheres) canalso be used and can in turn be attached to another solid support byknown means. The microspheres can be of any suitable size, typically inthe range of from 10 nm to 100 nm in diameter. In a preferredembodiment, the polynucleotides are attached directly onto a planarsurface, preferably a planar glass surface. Attachment will preferablybe by means of a covalent linkage. Preferably, the arrays that are usedare single molecule arrays that comprise polynucleotides in distinctoptically resolvable areas, e.g., as disclosed in International App. No.WO 00/06770.

The sequencing method can be carried out on both single polynucleotidemolecule and multi-polynucleotide molecule arrays, i.e., arrays ofdistinct individual polynucleotide molecules and arrays of distinctregions comprising multiple copies of one individual polynucleotidemolecule. Single molecule arrays allow each individual polynucleotide tobe resolved separately. The use of single molecule arrays is preferred.Sequencing single molecule arrays non-destructively allows a spatiallyaddressable array to be formed.

The method makes use of the polymerisation reaction to generate thecomplementary sequence of the target. The conditions necessary forpolymerisation to occur will be apparent to the skilled person.

To carry out the polymerase reaction it will usually be necessary tofirst anneal a primer sequence to the target polynucleotide, the primersequence being recognised by the polymerase enzyme and acting as aninitiation site for the subsequent extension of the complementarystrand. The primer sequence may be added as a separate component withrespect to the target polynucleotide. Alternatively, the primer and thetarget polynucleotide may each be part of one single stranded molecule,with the primer portion forming an intramolecular duplex with a part ofthe target, i.e., a hairpin loop structure. This structure may beimmobilised to the solid support at any point on the molecule. Otherconditions necessary for carrying out the polymerase reaction, includingtemperature, pH, buffer compositions etc., will be apparent to thoseskilled in the art.

The modified nucleotides of the invention are then brought into contactwith the target polynucleotide, to allow polymerisation to occur. Thenucleotides may be added sequentially, i.e., separate addition of eachnucleotide type (A, T, G or C), or added together.

If they are added together, it is preferable for each nucleotide type tobe labelled with a different label.

This polymerisation step is allowed to proceed for a time sufficient toallow incorporation of a nucleotide.

Nucleotides that are not incorporated are then removed, for example, bysubjecting the array to a washing step, and detection of theincorporated labels may then be carried out.

Detection may be by conventional means, for example if the label is afluorescent moiety, detection of an incorporated base may be carried outby using a confocal scanning microscope to scan the surface of the arraywith a laser, to image a fluorophore bound directly to the incorporatedbase. Alternatively, a sensitive 2-D detector, such as a charge-coupleddetector (CCD), can be used to visualise the individual signalsgenerated. However, other techniques such as scanning near-field opticalmicroscopy (SNOM) are available and may be used when imaging densearrays. For example, using SNOM, individual polynucleotides may bedistinguished when separated by a distance of less than 100 nm, e.g., 10nm to 10 μm. For a description of scanning near-field opticalmicroscopy, see Moyer et al., Laser Focus World 29:10, 1993. Suitableapparatus used for imaging polynucleotide arrays are known and thetechnical set-up will be apparent to the skilled person.

After detection, the label may be removed using suitable conditions thatcleave the linker.

The use of the modified nucleotides is not limited to DNA sequencingtechniques, and other techniques, including polynucleotide synthesis,DNA hybridisation assays and single nucleotide polymorphism studies, mayalso be carried out using nucleotides of the invention. Any techniquethat involves the interaction between a nucleotide and an enzyme maymake use of the molecules of the invention. For example, the moleculemay be used as a substrate for a reverse transcriptase or terminaltransferase enzyme.

Suitable structures are described in the following Examples and areshown in the accompanying drawings.

EXAMPLES Example 1 Synthesis of Disulfide Linker

tButyl-N-(2-mercaptoethyl) carbamate (3 mmol, 0.5 mL) was added dropwiseto a solution of 1.32 g (6.0 mmol) aldrithiol in 15 mL MeOH. After 1.5 hthe reaction had gone to completion and the solvent was evaporated. Thecrude product was purified by chromatography on silica with ethylacetate:petroleum ether (1:4). Product 1a was obtained as a slightlyyellow oil (0.76 g, 2.67 mmol, 89%). ¹H NMR (500 Mhz, D₆-DMSO): δ=1.38(s, 9H, tBu), 2.88 (t, J=6.6 Hz, 2H, SCH₂) 3.20 (q, J=6.6 Hz, 2H, CH₂NH), 7.02 (bs, 1H, NH), 7.24 (ddd, J=7.3 Hz, J=4.9 Hz, J=1.0 Hz, 1H,H-5), 7.77 (dt, J=8.1 Hz, J=1.0 Hz, 1H, H-3), 7.82 (ddd, J=8.1 Hz, J=7.4Hz, J=1.8 Hz, 1H, H-4), 8.46 (ddd, J=4.9 Hz, J=1.8 Hz, J=1.0 Hz, 1H,H-6).

To deprotect the amine of 1a, 17 mg of 1a (60 μmol) was dissolved in amixture of 0.5 mL DCM and 0.5 mL trifluoracetic acid. This mixture wasstirred for 2.5 h at rt and then the solvents were removed under reducedpressure. The residue was three times redissolved in 2 mL DCM andevaporated to dryness. The deprotected product was dried under highvacuum for 3 h and then dissolved in 1 mL dry DMF. It was assumed thatthe deprotection had gone to completion.

To a solution of 15 mg 5-carboxy tetra methyl rhodamine (35 μmol) in 2mL DMF were added 8.0 mg N-hydroxy succinimide (70 μmol) and 7.8 mg DCC(38 μmol). The mixture was stirred for 6 h in the dark. Then 22 μl DIPEA(126 μmol) and the solution of deprotected 1a in 1 mL DMF were added.After stirring the reaction mixture overnight in the dark, the solventwas removed under reduced pressure. The residue was dissolved in DCM andwashed with saturated NaCl solution. After drying over MgSO₄ the crudemixture was purified on silica with CHCl₃:MeOH (3:1) as solvent. 1b wasisolated as a dark red solid in 90% yield (19.2 mg, 31.4 μmol). ¹H NMR(500 MHz, D₆-DMSO): δ=3.09 (t, J=6.7 Hz, 2H, SCH₂), 3.63 (q, J=6.2 Hz,2H, CH₂ NH), 6.48-6.53 (m, 6H, H-Anthracene), 7.23-7.26 [m, 1H, H-5(pyridine)], 7.32 (d, J=7.9 Hz, 1 Hz, H-3), 7.81-7.82 [m, 2H, 1′-3+H-4(pyridine)], 8.21 (d, J=7.9 Hz, 1H, H-4), 8.43 (s, 1H, H-6), 8.47 [dt,J=4.7 Hz, J=1.3 Hz, 1H, H-6 (pyridine)], 9.03 (t, J=5.2 Hz, 1H, NH).

Mercaptopropionic acid (20.6 mmol, 1.8 μl) was added to a solution of19.6 mg 1b (32.7 mmol) in 2 mL MeOH. The mixture was stirred for 2.5 hin the dark. The solvent was removed under reduced pressure. The crudeproduct was purified by chromatography on silica with CHCl₃:MeOH:AcOH15:1:0.5 as the solvent mixture. 15.5 mg (26 mmol, 80%) dark redcrystals 1c could be isolated. ¹HNMR (500 MHz, D₂O): δ=2.53 (t, J=7.0Hz, 2H, CH₂ COOH), 2.88 (t, J=7.0 Hz, 2H, CH₂ CH₂COOH), 2.96-2.99 (m,2H, CH₂ CH₂NH), 3.73 (t, J=6.3 Hz, 2H, H₂ NH), 6.53 (d, J=2.4 Hz, 2H,H-Anthracene), 6.81 (dd, J=9.5 Hz, J=4.5 Hz, 2H, H-Anthracene), 7.12 (d,J=9.5 Hz, 2H, H-Anthracene), 7.48 (d, J=7.9 Hz, 1H, H-3), 7.95 (dd,J=8.1 Hz, J=1.9 Hz, 1H, H-2) 8.13 (d, J=1.9 Hz, 1H, H-1). +ve electrospray (C₃₀H₃₁N₃O₆S₂): expected 593.17. found 594.3 [M+H], 616.2 [M+Na].

TO a solution of 25.8 mg 1c (43.4 μmol) in 3 mL DMF (dry) were added 9.9mg N-hydroxy succinimide (86.8 μmol) and 9.7 mg DCC (47.1 μmol). Themixture was stirred in the dark for 5 h at room temperature and then putin the fridge overnight. The mixture was filtered through a plug ofcotton wool in a new flask and to this was added a solution of 865propargylamino dUTP (14.7 μmol, 17 μmol in 1 mL H₂O) and 3 mL sodiumborate buffer (0.1 M solution, pH 9). The mixture was stirred overnight.After removal of solvents the residue was dissolved in as little wateras possible and purified by HPLC. A Zorbax C18 column was used with 0.1M triethyl ammonium bicarbonate (TEAB) and acetonitrile as buffers. ³¹PNMR (400 MHz, D₂O): δ=−4.73 (d), −9.93 (d), 19.03 (t). −ve electro spray(C₄₂H₄₇N₆O₁₉P₃S₂ assuming 4H⁺ counter ions): expected 1096.16. found1092.9. UV in Water: λ_((max))=555 nm A₍₅₅₅₎=0.885 (c=0.036 μmol).

Triphosphate (1) was successfully incorporated using Klenow DNApolymerase. The reaction was performed in the following conditions: 50mM Tris.HCl (pH 7.5), 10 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 5 mM MgCl₂, 2μM compound 3, 100 nM DNA template (previously labelled with P32 and T4polynucleotide kinase) and 10 units of commercial exo-Klenow (AmershamCorp., Arlington Heights, Ill., USA). The DNA templates wereself-complementary hairpins(5′-TACCgTCgACgTCgACgCTggCg-AgCgTgCTgCggTTTTT(C6-amino)TTACCgCAgCACgCTCgCCAgCg;SEQ ID NO:1). The reaction was performed in 100 μL volume at 37° C. withtimepoints taken at 0, 1, 3, 5 and 10 min. The reaction products wereelectrophoresed down a denaturing (8 M urea) 20% polyacrylamide gel andimaged on a typhoon phosphorimager. Complete single base extension wasseen in 1 minute indicating efficient polymerase incorporation(disulfide linker gel, FIG. 3). A second set of lanes is shown in whichthe material is exposed to DTT after the incorporation. A different bandshift can be seen which shows removal of the dye from the DNA construct,thus a cycle of polymerase incorporation and cleavage has been shownusing this disulfide compound.

Example 2 Synthesis of TMR-Sieber Linker Free Acid

5-[-9-[9-(fluorenyl-methyloxycarbonyl)amino]xanthen-3-yl]valeric acid,(42.8 mg, 80 μmol) was stirred at room temperature with disuccinimidylcarbonate (22.5 mg, 88 μmol) and N,N-dimethyl aminopyridine (10.8 mg, 88μmol) in DMF. After 5 minutes, mono-5-carboxy TMR ethylene diamine(198.9 mg, 40 μmol) was added followed by DIPEA (13.9 μl, 80 μmol). Thereaction was stirred at room temperature. After 2 hrs, the reactionmixture was diluted with dichloromethane (100 mL) and the resultingsolution was extracted with 1 M aqueous potassium dihydrogen phosphate(50 mL). The DCM layer was separated and evaporated under reducedpressure. The residue was purified by a short column chromatography. Thefractions eluting with 40% methanol in chloroform were collected andevaporated under reduced pressure. The residue was then dissolved in dryDMF (1 mL) and N-(2-mercaptoethyl)aminomethyl polystyrene (200 mg, 400μmol) and DBU (12 μl, 80 μmol). After 10 minutes at room temperature,the resins were filtered off and rinsed with dry DMF (1 mL). All thefiltrates were combined and then added to a solution of succinicanhydride (80 mg, 800 μmol), DIPEA (139 μl, 800 μmol) and DMAP (9.8 mg,80 μmol) in DMF (1 mL). The reaction mixture was then stirred at roomtemperature. After overnight (16 hrs), all the solvents were evaporatedunder reduced pressure and the residue was purified by a short columnchromatography. The title compound eluted with 30% methanol inchloroform was obtained as purple powders (22 mg, overall yield 63%).¹HNMR [D₆-DMSO]: 8.82 (1H, t, J5.4, ex.), 8.75 (1H, d, J8.9, ex.), 8.42(1H, d, J 1.5), 8.20 (1H, dd, J 8.0 and 1.5), 7.95 (1H, t, J5.9, ex.),7.34 (1H, d, J7.3), 7.30-7.27 (2H, m), 7.21 (1H, d, J 8.5), 7.16-7.07(2H, m), 6.68 (1H, dd, J8.8 and 2.5), 6.65 (1H, d, J2.4), 6.49-6.43 (6H,m), 6.18 (1H, d, J5.6), 3.95 (1H, t, J5.9), 3.39-3.36 (2H, m), 3.30-3.27(2H, m), 2.92 (12H, s), 2.37-2.33 (2H, m), 2.14 (2H, t, J 7.2) and1.70-1.62 (4H, m). MS[(ES(+)], m/z 868.5 (MH⁺).

Example 3 Synthesis of TMR-Sieber Linker-dUTP (3)

TMR-Sieber linker free acid (4.34 mg, 5 μmol) was stirred withdisuccinimidyl carbonate (1.74 mg, 7.5 μmol) and N,N-dimethylaminopyridine (0.92 mg, 7.5 μmol) in DMF (1 mL) at room temperature.After 10 minutes, all the reaction mixture was added totetra-(tri-butylammonium) salt of5-(3-aminopropynyl)-2′-deoxyuridine-5′-triphosphate (10 μmol). Thereaction was stirred at room temperature for 4 hrs and stored in thefridge overnight. The reaction mixture was then diluted with chilledwater (10 mL) and all the resulting solution was applied onto a shortcolumn of DEAF A-25. The column was initially eluted with 0.1 M TEABbuffer and then 0.7 M TEAB buffer. The 0.7 M TEAB eluents were collectedand evaporated under reduced pressure. The residue was co-evaporatedwith MeOH (2×10 mL) and then purified by preparative HPLC. The titlecompound was obtained as triethylammonium salt in 31% yield (based onthe quantification of TMR at 555 nm in water (pH 7)). ¹HNMR in D₂Oindicated two diastereoisomers, due to the sieber linker moiety andthere were approximately three triethylammonium count ions. ¹HNMR [D₂O]:8.18 (1H, m), 8.06 (1H, m), 7.76 (0.55H, s), 7.74 (0.45H, s), 7.36-7.09(5H, m), 6.89-6.72 (3H, m), 6.59-6.37 (5H, m), 6.12 (0.5511, t, J6.6),6.05 (0.45H, t, J6.6), 5.99 (0.45H, d, J 2.5), 5.91 (1.1H, m), 5.88(0.45H, s), 4.49 (0.55H, m), 4.43 (0.45H, m), 4.00-3.35 (9H, m),3.30-2.95 (32H, m), 2.65-2.52 (4H, m), 2.25-2.05 (4H, m), 1.62-1.42 (4H,m) and 1.23 (27H, t, J 7.3). ³¹P [D₂O]: −9.91 (^(γ)P, d, J 19.2),[−11.08 (^(α)P, d, J 20.1) and −11.30 (^(α)P, d, J 20.1), due to twodiastereoisomers] and −22.57 (^(β)P, m). MS[(ES(−)], m/z 1369.1 (M⁻).

Triphosphate (3) was successfully incorporated using Klenow DNApolymerase. The reaction was performed in the following conditions: 50mM Tris.HCl (pH 7.5), 10 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 5 mM MgCl₂, 2μM compound 3, 100 nM DNA template (previously labelled with P32 and T4polynucleotide kinase) and 10 units of commercial exo-Klenow (AmershamCorp. Arlington Heights, Ill., USA). The DNA templates wereself-complementary hairpins(5′-TACCgTCgACgTCgACgCTggCg-AgCgTgCTgCggTTTTT(C6-amino)TTACCgCAgCACgCTCgCCAgCg;SEQ ID NO:1). The reaction was performed in 100 μL volume at 37° C. withtimepoints taken at 0, 1, 3, 5 and 10 min. The reaction products wereelectrophoresed down a denaturing (8 M urea) 20% polyacrylamide gel andimaged on a typhoon phosphorimager. Complete single base extension wasseen in 1 minute indicating efficient polymerase incorporation (Sieberlinker gel, FIG. 4).

Example 4 Synthesis of TMR-Indole Linker-dUTP (4)

Triphosphate (4) was successfully incorporated using Klenow DNApolymerase. The reaction was performed in the following conditions: 50mM Tris.HCl (pH 7.5), 10 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 5 mM MgCl₂, 2μM compound 3, 100 nM DNA template (previously labelled with P32 and T4polynucleotide kinase) and 10 units of commercial exo-Klenow (AmershamCorp., Arlington Heights, Ill., USA). The DNA templates wereself-complementary hairpins(5′-TACCgTCgACgTCgACgCTggCg-AgCgTgCTgCggTTTTT(C6-amino)TTACCgCAgCACgCTCgCCAgCg;SEQ ID NO:1). The reaction was performed in 1004 volume at 37° C. withtimepoints taken at 0, 1, 3, 5 and 10 min. The reaction products wereelectrophoresed down a denaturing (8 M urea) 20% polyacrylamide gel andimaged on a typhoon phosphorimager. Complete single base extension wasseen in 1 minute indicating efficient polymerase incorporation (indolelinker gel, FIG. 5).

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

What is claimed is:
 1. A method for detecting the extension of a primerannealed to a target polynucleotide, comprising: (a) providing at leastone nucleoside comprising (1) a label attached via a first linker to thenucleoside base, and (2) a protecting group attached via a second linkerto the 2′ or 3′ oxygen of the nucleoside sugar, wherein the first andsecond linkers are both cleavable under a single set of conditions; (b)extending the annealed primer with the nucleoside only if the nucleosidebase is complementary to the base at the corresponding position of thetarget polynucleotide; (c) detecting the presence of the label on theextended primer; and (d) incubating the extended primer under the singleset of cleavage conditions, whereby both the first and second linkersare cleaved; thereby detecting the extension of the primer.
 2. Themethod of claim 1, wherein the target polynucleotide is immobilized. 3.The method of claim 1, wherein step (a) provides at least two differentnucleosides, each having a different label, and the different labels aredistinguishable in step (c).
 4. The method of claim 3, wherein step (a)provides four different nucleosides.
 5. The method of claim 1, whereinthe nucleoside provided in step (a) is phosphorylated as a nucleotide.6. The method of claim 5, wherein the nucleotide provided in step (a)comprises three phosphate groups.
 7. The method of claim 1, wherein thefirst and/or second linkers comprise an azide.
 8. The method of claim 1,wherein the first and/or second linkers comprise an allylic group. 9.The method of claim 1, wherein extending step (b) is performed by a DNApolymerase.
 10. The method of claim 1, wherein extending step (b) isperformed by a reverse transcriptase.
 11. The method of claim 1, whereinextending step (b) is performed by a terminal transferase.
 12. Themethod of claim 1, wherein after step (b), any non-incorporatednucleosides are separated from the extended primers.
 13. The method ofclaim 1, wherein step (d) removes the protecting group to expose ahydroxyl.
 14. The method of claim 1, further comprising: (e) repeatingsteps (a), (b), (c), and (d) sequentially.
 15. The method of claim 14,wherein step (e) is repeated more than once.
 16. The method of claim 14,wherein steps (a)-(d) are initially performed with one nucleoside andsteps (a)-(d) in step (e) are performed with a different nucleoside. 17.The method of claim 16, wherein two nucleosides are used initially, andtwo different nucleosides are used in step (e).